Abstract
Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca2+-sensitive activation of retinal guanylyl cyclase 1 (RetGC1) upon light activation of photoreceptor cells. Here we present NMR assignments and functional analysis to probe Ca2+-dependent structural changes in GCAP1 that control activation of RetGC. NMR assignments were obtained for both the Ca2+-saturated inhibitory state of GCAP1 versus a GCAP1 mutant (D144N/D148G, called EF4mut), which lacks Ca2+ binding in EF-hand 4 and models the Ca2+-free/Mg2+-bound activator state of GCAP1. NMR chemical shifts of backbone resonances for Ca2+-saturated wild type GCAP1 are overall similar to those of EF4mut, suggesting a similar main chain structure for assigned residues in both the Ca2+-free activator and Ca2+-bound inhibitor states. This contrasts with large Ca2+-induced chemical shift differences and hence dramatic structural changes seen for other NCS proteins including recoverin and NCS-1. The largest chemical shift differences between GCAP1 and EF4mut are seen for residues in EF4 (S141, K142, V145, N146, G147, G149, E150, L153, E154, M157, E158, Q161, L166), but mutagenesis of EF4 residues (F140A, K142D, L153R, L166R) had little effect on RetGC1 activation. A few GCAP1 residues in EF-hand 1 (K23, T27, G32) also show large chemical shift differences, and two of the mutations (K23D and G32N) each decrease the activation of RetGC, consistent with a functional conformational change in EF1. GCAP1 residues at the domain interface (V77, A78, L82) have NMR resonances that are exchange broadened, suggesting these residues may be conformationally dynamic, consistent with previous studies showing these residues are in a region essential for activating RetGC1.
Highlights
Guanylyl cyclase activating proteins (GCAPs) belong to the neuronal calcium sensor (NCS) branch of the calmodulin superfamily [1,2,3] and regulate Ca2+-sensitive activity of retinal guanylyl cyclase (RetGC) in rod and cone cells [4,5,6]
A major difference in the structural dynamics between the different NCS proteins is the lack of a Ca2+myristoyl switch in GCAP proteins [28,44,45], and different structural roles for myristoylation and metal binding to control membrane targeting [46,47]
The NMR chemical shift differences between Ca2+-saturated wild type Guanylyl cyclase activating protein 1 (GCAP1) and EF4mut are relatively small (Figure 4), suggesting a similar overall main chain conformation for the two conformational states of GCAP1. This is in a sharp contrast to large Ca2+-induced conformational changes seen for the corresponding residues in other NCS proteins, including recoverin [23] and NCS-1 [29]
Summary
Guanylyl cyclase activating proteins (GCAPs) belong to the neuronal calcium sensor (NCS) branch of the calmodulin superfamily [1,2,3] and regulate Ca2+-sensitive activity of retinal guanylyl cyclase (RetGC) in rod and cone cells [4,5,6]. Light-activated channel closure blocks the entry of Ca2+, which lowers the cytosolic Ca2+ concentration from ~250-500 nM in the dark down to ~25 nM in the light [11]. This drop in Ca2+ causes the change in formation of Ca2+-free/Mg2+-bound GCAPs that activate RetGC [12], whereas Ca2+-bound GCAPs inhibit RetGC at high Ca2+ levels maintained in the dark [13,14,15]. The structure of the physiological activator form of GCAPs (Mg2+bound/Ca2+-free state) is currently unknown
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